Subunit exchange chromatography is a bioaffinity method that allows the purification of oligomeric and self-associating protein systems by means of their subunits immobilized on a solid matrix.
The method is based on the specificity of subunit recognition and exploits the observation that properly immobilized subunits retain the capacity to interact in a reversible and specific way with soluble subunits of the same or of homologous proteins. The experimental conditions that cause the establishment of a finite association-dissociation equilibrium in solution will promote the establishment also of a new equilibrium between immobilized and soluble subunits. The subunits will be exchanged between the liquid and the solid phase, and part of the protein that was initially in solution will be bound to the matrix. The amount of matrix-bound oligomer is a function of several parameters, i.e. concentrations of immobilized and soluble protein, and the association constants in solution and in the solid phase.
The possibility of shifting the subunit association-dissociation equilibrium by changing the experimental conditions enables one to achieve an effective purification of a self-associating or oligomeric protein in two simple steps. First, the protein is extracted, for example, from a tissue homogenate or the culture medium and bound to its highly purified subunits or subunits of homologous protein immobilized under conditions that favor binding. Second, the protein is dissociated by eluting under conditions that favor the dissociation, without destroying the biological activity of the eluted protein and the immobilized subunits. The immobilized subunits are regenerated and become ready for a new purification cycle after equilibration with the associating buffer. It is best to immobilize the protein under conditions that stabilize the subunit. In this way, coupling to the resin through residues that are located at or near the intersubunit contact regions is avoided and immobilized subunits that display minimal heterogenecity in their interaction with soluble subunits are generated. A variety of systems have been purified successfully by means of immobilized subunits of the same protein or of homogolous subunits of the same protein capable of forming a hybrid with the protein to be isolated.
Additional information about this chromatographic method is reported by Chiancone E; et al., J. Chromatography 376, 343-348 (1986), Antonini E. et al., Anal. Biochem. 95, 89-96 (979), Carrea G., FEBS Letters 104, 393-395 (1979).
The present invention utilizes the basic concepts of such subunit exchange chromatography, with important improvements to prepare highly purified BNGF.